HCV is a causal factor of chronic hepatitis C, and, according to statistics reported by WHO, it is assumed that there are 170 million infected patients in the world. HCV is a virus classified into the genus Flavivirus in the family Flaviviridae. It is considered that infection with the virus occurs via blood or a blood component, and the virus grows in liver. In the initial phase of infection, patients infected with HCV show only mild symptoms, but the infection becomes chronic at high frequency, leading, after a certain length of asymptomatic period, to development of chronic hepatitis. As the infection continues, exacerbation of the disease condition occurs to cause liver cirrhosis, which then leads to liver cancer at high frequency. It is considered that hepatitis virus is involved in 95% of liver cancer, and that infection with HCV is responsible for 80% of such cases.
For treatment of chronic hepatitis C, interferon is widely used. In recent years, the rate of complete cure by elimination of HCV in vivo has been gradually increasing due to improved formulations of interferon and improved administration methods such as combination therapy with interferon and ribavirin. However, the complete cure rate by administration of interferon is still about 50%, and it is considered that many types of HCV are resistant to interferon therapy. Thus, development of a drug having a therapeutic effect against the interferon-resistant virus has been demanded.
Development of such a drug requires a drug screening system. Although a method by infecting cells derived from human or monkey with HCV in vitro and propagating HCV has been reported, such a propagation system could not be used as a drug screening system because of both low infection efficiency and low growth efficiency.
Wakita et al. isolated an HCV gene of genotype 2a from a fulminant hepatitis C patient (Patent Document 1). From the isolated JFH1 strain, full-length RNA was synthesized in vitro, and the RNA was transfected into human liver cancer-derived cells (Huh7 cells). As a result, a replicon RNA that autonomously replicates in the cells was successfully obtained. Further, release of infectious particles into the culture supernatant of the cells into which the replicon RNA was transfected was confirmed (Non-patent Document 1). Therefore, by transfecting the replicon RNA of the JFH1 strain into human liver cancer-derived cells (Huh7 cells) and culturing the obtained infectious particles again with human liver cancer-derived cells, a reinfection-propagation system can be constructed. By using this reinfection-propagation system, screening of drugs against HCV has been started.
However, the JFH1 strain is HCV of genotype 2a, which is an interferon-sensitive HCV. Therefore, since the strain does not have an HCV gene region responsible for resistance to interferon, host factors that act on the region providing resistance to interferon cannot be identified. Thus, screening of drugs effective against interferon-resistant HCV might be impossible.
Lemon et al. reported an infection-propagation system prepared by transfecting the replicon RNA of the H77 strain of genotype 1a into human liver cancer-derived cells (Huh7 cells) (Non-patent Document 2). However, when virus particles obtained from the culture supernatant of the cells having the transfected replicon RNA were used again for infection of the human liver cancer-derived cells, the infectivity titer was 400 times lower than that of infectious particles of the JFH1 strain. Therefore, it is considered that the replicon RNA of the H77 strain releases virus particles which lost the infectivity. Thus, it is considered that the replicon RNA of the H77 strain which can replicate in vitro has already lost the function to produce infectious particles and does not have the inherent growth function of HCV. Thus, screening of drugs effective against HCV having the function to grow in the living body might be impossible with a screening system using the infection-propagation system of the replicon RNA of the H77 strain.
Although, as described above, the replicon RNAs reported by Wakita and by Lemon partially enabled screening of drugs, those replicon RNAs have the above-described problems, and it is considered that screening of drugs that can be widely used for treatment of HCV is impossible with those replicon RNAs.
Further, for obtaining a drug that can be widely used for treatment of HCV, the present inventors also developed pTPF1/4B as an in vitro propagation system which has an efficient HCV propagation, gene of genotype 1b, resistance to interferon, and ability to produce infectious particles (Patent Document 2). However, this culture system was practically problematic in Huh7 cells, which are human liver cancer-derived cells, since the efficiencies of autonomous replication in the cell, self-replication of RNA and secretion of virus particles into the culture supernatant were poor. Further, in Non-patent Document 3, it is reported that there is a region, in the C-terminus side of the NS2 region, which is important for infectivity.